1 kb ladder

The single crypt lysate (see above) was used as the DNA template for long-range PCR to determine whether large-scale circular mtDNA deletions were present. Two rounds of PCR were carried out. PCR was performed using Takara LATak PCR system (Takara Bio Inc.) according to manufacturer's recommended conditions. After 30 cycles of first round PCR (95 °C for 20 s, 68 °C for 16 min), 2 μl of the first round product was used as template for the second round PCR reaction (same as above) and 20 further cycles of PCR carried out. First round PCR primers were L272–L301 and H16286–H16254; second round primers were L1275–L13004 and H15833–H15804 giving an expected product size of ∼14.5 kb PCR products were subjected to electrophoresis through a 0.8% agarose gel and the band sized against a 1 kb Ladder (Promega).

Molecular typing of the UEc isolates was performed using enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) according to specific modifications of the protocols established by Ardakani and Ranjbar [21 (link)]. ERIC-PCR was performed on 25 μL reaction volumes containing 1 μL of each primer (25 pmol), 12.5 μL of the master mix, 1.5 μL of MgCl₂ (300 nM), 1 μL of the DNA template (100 ng/μL), and 8 μL of deionized water. The ERIC-PCR protocol was divided into 3 steps: 1) denaturation at 94°C for 2 min; 2) 30 cycles of denaturation at 94°C for 1 min, annealing at 52°C for 1 min, and extension at 72°C for 4 min; and 3) a final extension at 72°C for 5 min. PCR products were resolved on 2% agarose gels (Promega Corporation, WI, USA) by electrophoresis at 80 volts for 120 min. The PCR products resolved on the gels were stained with 0.5 mg/mL ethidium bromide (Sigma Aldrich) for 40 min and visualized under UV light using a ChemiDoc MP imaging system. A 1 kb ladder and a 100 bp ladder (Promega Corporation; WI, USA) were used as molecular weight markers.

Genomic DNA extraction of P. yayanosii CH1 was performed using a phenol–chloroform–isoamyl alcohol (PCI) method as described by Thiel et al.49 (link). The DNA was quantified by NanoDrop and the quality of extraction was checked by electrophoresis on a 1% agarose gel containing ethidium bromide at a final concentration of 0.5 mg/ml (in a bath of 40 mM Tris pH 8, 40 mM acetate-1 mM EDTA pH 8 (TAE) 1X). The migration was performed at 85 V for 40 min, with a 1 kb ladder (Promega) as the size marker. The routine tests by PCR amplification were performed using Taq Polymerase (Promega).